A Spot Plate Assay for Evaluating Oxidative Stress Responses in Bacterial Strains

Treat both pyomelanin and non-pyomelanin-producing bacterial strains with a test agent. 

Pyomelanin, an extracellular pigment, provides protection against oxidative stress.

The test agent disrupts pyomelanin biosynthesis, reducing its production.

Post-incubation, perform serial bacterial dilutions with a buffer containing the test agent or vehicle solvent.

Spot bacterial dilutions on agar plates with varying hydrogen peroxide concentrations — an oxidative stressor.

Plates lacking hydrogen peroxide serve as controls.

Incubate. In control plates, bacteria form colonies.

In the untreated pyomelanin-producing strain, pyomelanin shields bacteria from hydrogen peroxide-induced oxidative damage, resulting in pigmented colonies.

However, the pyomelanin-producing strain treated with the test agent, exhibiting reduced pyomelanin, becomes sensitive to oxidative stress, resulting in bacterial death.

Count colonies.

The reduced colony count and size with increasing hydrogen peroxide concentrations in treated pyomelanin-producing and both treated and untreated non-pyomelanin-producing strains than controls indicate heightened bacterial sensitivity to oxidative stress.